The glandular kallikreins are a subgroup of serine proteases which are involved in the post-translational processing of specific polypeptide precursors to their biologically active forms. In humans, three members of this family have been identified, and some of their properties characterized (Clements, Endoc. Rev., 10, 343 (1989); Clements, Mol. Cell Endo., 99, 1 (1994); Jones et al., Acta Endoc., 127, 481 (1992)). The hKLK1 gene encodes the tissue kallikrein protein, hK1, the hKLK2 gene encodes the prostate-specific glandular kallikrein protein, hK2, and the hKLK3 gene encodes the prostate-specific antigen protein, hK3 (PSA). Northern blot analysis of mRNA shows that both hK2 and PSA are expressed mainly in the human prostate, while expression of hK1 is found in the pancreas, submandibular gland, kidney, and other nonprostate tissues (Chapdelaine et al., FEBS Lett., 236, 205 (1988); Young et al., Biochem. 31, 818 (1992)).
The nucleotide sequence homology between the exons of hKLK2 and hKLK3 is 80%, whereas the nucleotide sequence homology between the exons of hKLK2 and hKLK1 is 65%. The deduced amino acid sequence homology of hK2 to PSA is 78%, whereas the deduced amino acid sequence homology of hK2 to hK1 is 57%. Moreover, the deduced amino acid sequence of hK2 suggests that hK2 may be a trypsin-like protease, whereas PSA is a chymotrypsin-like protease.
PSA levels are widely used as a prognostic indicator of prostate carcinoma. However, since the concentration of PSA in serum is elevated in patients with either prostatic cancer (pCa) or benign prostatic hyperplasia (BPH), the detection of elevated levels of PSA does not distinguish between these diseases. Moreover, the high degree of homology of hK2 to PSA raises some question as to the specificity of antibodies currently used to detect the levels of PSA. If the levels of circulating hK2 are unrelated to pCa or BPH, then antibodies raised to preparations of PSA which are contaminated with hK2, or to regions of PSA with homology to hK2, can result in false positive results.
Although it is now generally accepted that serum PSA testing, combined with the digital rectal exam (DRE), is the most effective means to detect clinically significant and organ-confined prostate cancer, combinations of PSA, DRE and ultrasonic prostate examination can detect only some prostate tumors. For example, up to 40% of surgically treated patients with prostate cancer are subsequently found to be clinically understaged. Moreover, the actual incidence of histological cancers based on autopsy data relative to the incidence of clinically significant prostate cancer is high. Furthermore, approximately 30% of patients with alleged localized prostate cancer may have occult (distant) metastatic disease (Moreno et al., Cancer Res., 52, 6110 (1992)). Of these patients, 80% experience relapse biochemically, i.e., elevated PSA levels, or by the recurrence of local, or occurrence of frank systemic, disease, after therapy.
Operative therapy is not the appropriate treatment modality for patients having established metastasis. Screening modalities to assess early metastases often fail to identify a significant subset of patients with locally invasive disease involving penetration of the prostate capsule or seminal vesicle. While immunohistochemical techniques have been employed to identify micrometastatic, or circulating, prostate tumor cells when no obvious metastatic deposit was evidenced by conventional means, immunohistochemical methods are laborious and lack the sensitivity needed for the early detection of metastatic or locally invasive prostate cancer.
There is, therefore, a need for early detection of prostate cancer cells with metastatic potential. Moreover, there is a need to accurately stage prostate cancer prior to subjecting a patient to invasive procedures. In particular, there is a need for a marker for prostate cancer that can function independently of, or in combination with, PSA.
The invention provides a diagnostic method for detecting hK2 DNA wherein the presence of prostate cancer cells in a physiological sample can be correlated to the detection of hK2 RNA in the sample. Because expression of hK2 is prostate tissue specific, hK2 RNA should theoretically not be detectable in cells present in bodily fluids or non-prostate tissue if there is no locally invasive or metastatic disease, or if all prostate tissue (benign and malignant) has been removed or destroyed. The method comprises contacting an amount of DNA obtained by reverse transcription (RT) of RNA from a human physiological sample with a plurality of oligonucleotide primers, preferably at least two oligonucleotide primers, at least one of which an hK2-specific oligonucleotide, in an amplification reaction so as to yield an amount of amplified hK2 DNA. A preferred amplification reaction is a polymerase chain reaction (PCR). The presence of the amplified hK2 DNA is then detected. Preferably, the conditions are effective to amplify the amount of DNA obtained by reverse transcription of RNA from at least one cell containing hK2 in a sample which comprises at least about 107 to about 109 cells. As described hereinbelow, the presence of amplified hK2 DNA in blood cells, after RT-PCR, is correlated with prostate cancer, i.e., sixty-seven percent (67%) of the prostate cancer patients expressed hK2, 17% expressed PSA, and 17% expressed both hK2 and PSA. Preferably, the source of the sample to be tested is human tissue, e.g., prostate, prostate capsule, seminal vesicle, bone marrow or lymph node. Another preferred source of the sample to be tested is a human physiological fluid which comprises cells, e.g., blood, serum, or seminal fluid.
As used herein, xe2x80x9camplified hK2 DNAxe2x80x9d is defined to mean hK2 DNA in a sample, which was subjected to an amplification reaction, that is present in an amount that is greater than, i.e., 10, preferably 104, and more preferably 106, times greater than, the amount of hK2 DNA which was present in the sample prior to amplification.
As used herein, the term xe2x80x9chK2-specific oligonucleotidexe2x80x9d or xe2x80x9chK2-specific primerxe2x80x9d means a DNA sequence that has at least about 80%, more preferably at least about 90%, and more preferably at least about 95%, sequence identity or homology to SEQ ID NO:4 in regions of SEQ ID NO:4 that are divergent from nucleotide sequences which encode hK3 (SEQ ID NO:23). An oligonucleotide or primer of the invention has at least about 7-50, preferably at least about 10-40, and more preferably at least about 15-35, nucleotides. Preferably, the oligonucleotide primers of the invention comprise at least 7 nucleotides at the 3xe2x80x2 of the oligonucleotide primer which have at least about 80%, more preferably at least about 85%, and more preferably at least about 90%, identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The oligonucleotides of the invention may also include sequences which are unrelated to hK2 nucleic acid sequences, e.g., they may encode restriction endonuclease recognition sequences. A preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:14. Another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:17. Yet another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:18.
A preferred diagnostic method of the invention combines RT-PCR detection of hK2 transcripts with RT-PCR detection of transcripts of other gene products associated with prostate cancer. Combined detection of two or more gene products may provide greater diagnostic certainty or yield more informative staging information. Combined detection may also be helpful in differentiating those cells with aggressive growth potential from those that are more indolent. In a particularly preferred embodiment of the method provided by the invention, RT-PCR detection of hK2 RNA is combined with RT-PCR detection of PSA RNA.
The invention further provides a diagnostic method for detecting hK2 RNA. The method comprises extracting RNA from a physiological sample obtained from a human. The extracted RNA is reverse transcribed to yield DNA. The DNA is contacted with an amount of at least two oligonucleotides effective to amplify the DNA to yield an amount amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide. The presence of the amplified hK2 DNA is then detected. The presence of the amplified hK2 DNA is indicative of metastatic prostate cancer in the human. Preferably, the conditions are effective to amplify the amount of DNA obtained by reverse transcription of RNA from at least one cell containing hK2 in a sample which comprises at least about 107 to about 109 cells.
The presence of hK2 RNA, or a level of hK2 RNA that rises over time, in bodily fluids or non-prostate tissue may be reasonably expected to indicate the presence of previously undiagnosed metastatic disease. Early detection of metastatic disease provides a xe2x80x9clead timexe2x80x9d during which alternative therapeutic strategies, including those that may not exist at the time of surgery but are subsequently developed, can be evaluated. Thus, the present invention provides a method for monitoring the progression of prostate cancer.
The method comprises contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from a human afflicted with prostate cancer with an amount of at least two oligonucleotides, at least one of which an hK2-specific oligonucleotide, effective to amplify the DNA to yield an amount of amplified hK2 DNA. The presence or amount of the amplified hK2 DNA is detected or determined. At least one point later in time, another sample is taken and the amount of amplified hK2 DNA detected or determined. Then the amounts of amplified hK2 DNA, obtained at least at two different time points, are compared.
Also provided is a method for pathologically staging prostate cancer in a human. The method comprises contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from the human afflicted with prostate cancer with an amount of at least two oligonucleotides, at least one of which is an hK2-specific oligonucleotide, effective to amplify the DNA to yield an amount of amplified hK2 DNA. The presence or amount of the amplified hK2 DNA is then detected or determined. The presence or amount of amplified hK2 DNA is indicative of the pathological stage of the prostate cancer. Preferably, the conditions are effective to amplify the amount of DNA obtained by reverse transcription of RNA from at least one cell containing hK2 in a sample which comprises at least about 107 to about 109 cells.
Another embodiment of the invention provides a method for monitoring therapeutic interventions involving hormone therapies. For example, because hK2 expression is androgen-dependent, hK2 RNA levels in peripheral blood or other bodily tissue or fluid may be used as a marker during intermittent androgen therapy, or during androgen provocative testing, wherein a patient is temporarily placed in a hyperandrogenized state to stimulate a level of hK2 production by any persistent prostate cancer cells sufficient to render them detectable. See T. K. Takayama et al., Sem. in Oncol., 21, 542-553 (1994), and references cited therein, which are incorporated herein by reference. hK2 levels are preferably monitored periodically during the course of hormone therapy. It may be advantageous to also determine hK2 levels before commencement of therapy, and periodically after the conclusion of a therapeutic regimen.
Also provided is a diagnostic kit for detecting hK2 RNA in a physiological sample suspected of containing hK2 RNA. The kit comprises packaging containing (a) a known amount of a first hK2-specific oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to SEQ ID NO: 4 or SEQ ID NO:33, and (b) a known amount of a second hK2-specific oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to a nucleotide sequence which is complementary to SEQ ID NO:4 or SEQ ID NO:33. Thus, the invention also provides hK2-specific oligonucleotides consisting of at least about 7-50 nucleotides, wherein he oligonucleotide has at least about 80% identity to a nucleotide sequence which has at least 80% identity, or complementary, to SEQ ID NO:4 or SEQ ID NO:33. Preferred oligonucleotides useful in the practice of the invention comprise SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:27 and SEQ ID NO:28.
The invention further provides an isolated, purified peptide comprising SEQ ID NO:22, a biologically active subunit thereof, or a biologically active variant thereof. The invention further provides an isolated, purified peptide comprising SEQ ID NO:26, a biologically active subunit thereof, or a biologically active variant thereof. Also provided is an isolated purified antibody or antibody preparation that specifically reacts with a protein or polypeptide which comprises the peptides of the invention described above.
As used herein, the term xe2x80x9cbiologically active subunitxe2x80x9d of a peptide of the invention is preferably defined to mean a subunit of a peptide having SEQ ID NO:22, which has at least about 10%, preferably at least about 50%, and more preferably at least about 90%, the activity of a peptide having SEQ ID NO:22. The activity of a peptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the peptide to elicit a sequence-specific immunologic response when the peptide is administered to an organism, e.g., goat, rabbit, sheep or mice.
As used herein, the term xe2x80x9cbiologically active variantxe2x80x9d of a peptide of the invention is preferably defined to mean a peptide which has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, identity or homology to SEQ ID NO:22. Biologically active variants of the peptides of the invention have at least about 10%, preferably at least about 50%, and more preferably at least about 90%, the activity of a peptide having SEQ ID NO:22. The activity of a variant peptide of the invention can be measured by methods described hereinabove.
The invention further provides a method for detecting or determining the presence of metastatic prostate cancer in a human non-prostate tissue sample. The method comprises mixing an amount of an agent, which binds to an hK2 polypeptide and which does not bind to hK3, with the cells of the mammalian tissue sample so as to form a binary complex comprising the agent and the cells. The presence or amount of complex formation in the sample is then detected or determined. The presence or amount of the complex provides an indication of the presence of micrometastatic prostate cancer. As used herein, xe2x80x9cmicrometastaticxe2x80x9d means locally invasive disease, which typically involves penetration of the prostate capsule or seminal vesicle, or occult disease. A preferred agent for use in the method is an antibody. The term xe2x80x9cantibodyxe2x80x9d includes human and animal mAbs, and preparations of polyclonal antibodies, as well as antibody fragments, synthetic antibodies, including recombinant antibodies, chimeric antibodies, including humanized antibodies, anti-idiotopic antibodies and derivatives thereof. To prepare antibodies which bind to hK2 and not to hK3, isolated hK2 polypeptides, isolated hK2 peptides, as well as variants and subunits thereof, can be used to prepare populations of antibodies. These antibodies in turn can be used as the basis for direct or competitive assays to detect and quantify hK2 polypeptides (or xe2x80x9cproteinxe2x80x9d) in samples derived from tissues such as bone marrow and lymph nodes, and samples of cells such as from physiological fluids which comprise cells.